The magnitude of the AIDS epidemic has fueled research on the mechanisms involved in HIV-1 infection and pathogenesis. Discovery of the role of (chemokine) coreceptors in viral entry and the crystal structure on the gp120 subunit of the envelope glycoprotein (env) have opened critical avenues for studying the inception of HIV-1 infection. The V3 loop of gp 120 governs viral tropism through coreceptor usage CCR5 is used by env of commonly transmitted strains and CXCR4 by T-tropic strains that evolve in late phases of infection. After activation by CD4, gp 120 can interact with a coreceptor, thereby triggering membrane fusion and viral entry. The focus of the proposed research is to elucidate the mechanism of the interaction between gp120 and coreceptor, a critical point of attack to block HIV-1 infection. Whereas the N-terminus and hydrophobic core with interhelical loops of CCR5 are each sufficient to confer coreceptor activity to hybrids containing segments of inactive chemokine receptors, only that latter region of CXCR4 appears to be involved in the interaction with gpl20 We have demonstrated that gp120 subunits with V3 crown variant lack the ability to utilize the hydrophobic core and interhelical loops of CCR5 for env-mediated fusion and have impaired binding to the wild type receptor, suggesting that these two structures interact. The central hypothesis underlying the proposed research is that extracellular domains of the receptors transiently bind the V3 loop to induce a conformation that is permissive for further interactions involving the nascent coreceptor structures and conserved regions of gp120. The following specific aims are proposed to test this hypothesis 1) To characterize the role of CCR5 N-terminus and second extracellular loop domains in coreceptor activity, 2) To refine insight into the conformation of the CCR5 and CXCR4 hydrophobic core and interhelical loops involved in the interaction with antagonists of coreceptor activity, including ligands and antagonists, 3) To characterize the consequences of coreceptor signaling on the biology of HIV-1 replication using unique CCR5 and CXCR4 signaling variants generated by functionally coupling these coreceptors to the phermone pathway in yeast. The proposed experiments will combine the power of yeast genetics and molecular dynamic simulations to establish a structural basis that will guide the development of strategies to disrupt the role of frontline coreceptors in HIV-1 infection.